Generally, a pharmaceutical drug is subjected to animal experiments and clinical trials to sufficiently verify its efficacy and safety before approval. However, occasionally, after a pharmaceutical drug is put on the market, it exhibits unidentified fatal drug toxicity such as serious hepatotoxicity and allergic reactions. Such drug toxicity is referred to as idiosyncratic drug toxicity (IDT). At present, it is difficult to predict the occurrence of IDT, but it is considered that a “reactive metabolite” generated by metabolism of the drug is involved in the occurrence of IDT (Non Patent Literature 1). Therefore, it is important for the development of pharmaceutical drugs to devise a scheme for synthesizing drug candidate compounds that do not form reactive metabolites.
One known simple method for examining whether or not a drug candidate compound is metabolized to form a reactive metabolite is a trapping test using a trapping agent. A reactive metabolite is very unstable and is therefore difficult to detect. In the trapping test, the drug candidate compound is incubated in the presence of a metabolic enzyme to examine whether or not a reactive metabolite is formed. In this test, the trapping agent is coexisted with the drug candidate compound. The trapping agent bonds to a reactive metabolite formed from the drug candidate compound through the action of the metabolic enzyme to thereby form an adduct. This trapping agent-reactive metabolite adduct is relatively stable and can be detected using a mass spectrometer etc. Patent Literature 1 discloses glutathione as the trapping agent.
Other reported examples of the compound usable as the trapping agent include glutathione ethyl ester (Non Patent Literature 2). It has been reported that, when glutathione ethyl ester is used as the trapping agent in the analysis using a mass spectrometer, a trapping agent-reactive metabolite adduct can be detected with higher sensitivity than that when glutathione is used as the trapping agent (Non Patent Literature 2).
However, there is a fact that, when the above-described glutathione or glutathione ethyl ester is used as the trapping agent, it is difficult to identify the peak of a trapping agent-reactive metabolite adduct when a liquid chromatography-mass spectrometer (LC-MS) etc. is used to detect the trapping agent-reactive metabolite adduct. Therefore, a peak different from the peak of the trapping agent-reactive metabolite adduct may be misidentified as the peak of the trapping agent-reactive metabolite adduct, and this leads to a misjudgment that a reactive metabolite is formed (a false positive).
One means proposed to prevent a false positive is to use an isotope-labeled compound as a trapping agent. Examples of the isotope-labeled compound usable as the trapping agent include glutathione-glycine-13C2,15N (Patent Literature 2 and Non Patent Literatures 3 and 4). Glutathione-glycine-13C2,15N is an isotope-labeled compound in which each of two carbon atoms (12C) in the glycine moiety of glutathione is labeled with its isotope (13C) and one nitrogen atom (14N) in the glycine moiety is labeled with its isotope (15N). Glutathione-glycine-13C2,15N has a mass number larger by 3 than ordinary glutathione. When a mixture of glutathione-glycine-13C2,15N and ordinary glutathione at a certain ratio (e.g., 1:1) is used as a trapping agent, a glutathione-glycine-13C2,15N-reactive metabolite adduct and a glutathione-reactive metabolite adduct are formed at the above ratio. When an LC-MS, for example, is used to detect these adducts, isotope doublet peaks that differ by 3 in mass number appear. Therefore, the target peaks can be, easily distinguished, so that the possibility of a false positive result can be reduced.